Accepted Manuscript Title: Central metabolic pathways of Aureobasidium pullulans CGMCC1234 for pullulan production Author: Long Sheng Chang Liu Qunyi Tong Meihu Ma PII: DOI: Reference:
S0144-8617(15)00757-2 http://dx.doi.org/doi:10.1016/j.carbpol.2015.08.016 CARP 10219
To appear in: Received date: Revised date: Accepted date:
21-5-2015 4-8-2015 7-8-2015
Please cite this article as: Sheng, L., Liu, C., Tong, Q., and Ma, M.,Central metabolic pathways of Aureobasidium pullulans CGMCC1234 for pullulan production, Carbohydrate Polymers (2015), http://dx.doi.org/10.1016/j.carbpol.2015.08.016 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Central metabolic pathways of Aureobasidium pullulans CGMCC1234 for
Long Sheng a, Chang Liu b,c, Qunyi Tong c, Meihu Ma a, *
4 5 6
Huazhong Agricultural University, Wuhan, China
The State Key Laboratory of Food Science and Technology, Jiangnan University,
Ac ce pt e
Hsingwu Business and Tourism School, Shanghai Lida Polytechnic Institute,
National R&D Center for Egg Processing, College of Food Science and Technology,
*Corresponding author: Professor Meihu Ma
Tel: +86 27 87283177
Fax: +86 27 87283177
E-mail address: [email protected]
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Abstract With the purpose of understanding the metabolic network of Aureobasidium
pullulans, the central metabolic pathways were confirmed by the activities of the key
enzymes involved in different pathways. The effect of different iodoacetic acid
concentrations on pullulan fermentation was also investigated in this paper. The
activities of phosphofructokinases and glucose-6-phosphate dehydrogenase existed in
A. pullulans CGMCC1234, whereas 2-keto-3-deoxy-6-phosphogluconate aldolase
activity was not detected. We proposed that the central metabolic pathways of A.
pullulans CGMCC1234 included EMP and PPP, but no ED. Pullulan production
declined fast as the iodoacetic acid increased, while cell growth offered upgrade
firstly than descending latter tendency. Compared to the control group, the ratio of
ATP/ADP of 0.60 mM iodoacetic acid group was lower at different stages of pullulan
fermentation. The findings revealed that low concentration of iodoacetic acid might
impel carbon flux flow toward the PPP, but reduce the flux of the EMP.
Keywords: Aureobasidium pullulans; Pullulan; Central metabolic
Ac ce pt e
Aureobasidium pullulans is generally known as the strain producing pullulan, an
exocellular homopolysaccharide. Pullulan is broadly applied in the food and medical
industries because of its prominent chemical and physical properties, such as low
viscosity, nontoxicity, slow digestibility, high plasticity and excellent film-forming
(Cheng, Demirci, & Catchmark, 2011). Pullulan is a linear homopolysaccharide 2
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composed of maltotriose reduplicative units connected by α-1,4-linkages. This
particular linkage pattern confers pullulan with excellent solubility in water compared
to other polysaccharides (Jiang, Wu, & Kim, 2011). Pullulan biosynthesis is a complex metabolic process under the control of
environmental conditions. A. pullulans, commonly known as black yeast due to their
melanin production, is cosmopolitan yeast-like fungi (Gostincar et al., 2014). Owning
to the poor understand of the metabolic network of A. pullulans, the metabolic process
of A. pullulans could only be speculated by the similar characteristic of yeast in the
previous study (Shingel, 2004). However, A. pullulans has its unique metabolic
features and the cellular morphologies characteristics of A. pullulans are much more
luxuriant than yeast.
The central metabolic pathways are the foundation of all biological cell life
activities. It could transfer the nutrients to the structural unit to gratify own needs
(Lien et al., 2015). This process could supply precursor substance, Gibbs free energy
and reducing power for the cell’s physiological activities and product synthesis. The
central metabolic pathways generally consist of Embden Meyerhof Pathway (EMP),
Pentose Phosphate Pathway (PPP) and Entner-Doudoroff Pathway (ED).
Ac ce pt e
There are many reagents existing inhibition of glycolytic pathway, different
inhibitors could restrain different enzymatic reactions during the metabolic process.
The oxidation of glyceraldehyde 3-phosphate to glyceraldehydes-1, 2-diphosphate is
the sixth step of glycolytic pathway. It is the first time to produce ATP during
glycolytic pathway. This reaction is catalyzed by glyceraldehyde-3-phosphate 3
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dehydrogenase (GAPDH). GAPDH has four subunits, and every subunit has one
active site. Every active site of GAPDH contains a cysteine carrying with a free thiol
(-SH). Alkylating agent such as iodoacetic acid could form covalent binding of -SH,
and the inhibition is irreversible (Brooks & Storey, 1993). Therefore, iodoacetic acid
is always used as an inhibitor in glycolytic cycle.
In this research, the central metabolic pathways were elucidated. The effect of
different iodoacetic acid concentrations on pullulan fermentation was also studied.
The results are necessary for understanding the fungal metabolism regulation
2. Materials and methods
76 77 78 79
A. pullulans CGMCC1234 was conserved on potato dextrose agar (PDA) at 4°C
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and cultured every 2 weeks again.
2.2. Seed culture
The seed broth included 50.0 g sucrose, 4.0 g K2HPO4, 2.0 g NaCl, 1.5 g yeast
extract, 0.8 g (NH4)2SO4, and 0.2 g MgSO4 in 1 L distilled water. The medium pH
was adjusted to 6.5.
82 83 84
2.3. Culture conditions Cultures were grown on PDA at 28 °C for 4 days and then transferred to a 250 4
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mL flask that contained 50 mL of the seed culture medium and subsequently
incubated at 28 °C for 2 days with shaking at 200 rpm. A total of 2.5 ml of seed broth
was then inoculated into 250 mL flask containing 50 mL of the fermentation medium
and the flasks were incubated at 28 °C for 96 h on a rotary shaker at 200 rpm. The
fermentation medium containing (g/L) 80.0 g sucrose, 0.9 g yeast extract, 6.0 g
K2HPO4, 0.6 g (NH4)2SO4, 0.5 g MgSO4, 4.0 g NaCl and different concentrations of
2.4. Preparation of cell-free extract
Cell-free extract preparation was according to our previous work (Sheng, Zhu, &
Tong, 2013). Protein concentration in the cell-free extract was determined by the
method of Bradford with bovine serum albumin as standard (Bradford, 1976).
d Ac ce pt e
2.5. Determination of enzyme activity The measurement of enzyme activity for phosphofructokinases (PFK),
glucose-6-phosphate dehydrogenase (G6PD) and 2-keto-3-deoxy-6-phosphogluconate
aldolase (KDPG aldolase) was followed the method described previously (Grobben et
al., 1996; Karakaya et al., 2009; Griffiths et al., 2002). The enzyme activity was
expressed in nmol per min per mg protein.
104 105 106
2.6. Analytical methods The determination of pullulan production and biomass was according to our 5
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previous work (Sheng, Zhu, & Tong, 2014). ATP and ADP in the cell-free extract
were determined by high-performance liquid chromatography (HPLC) using a
reverse-phase ODS column (150 × 4.6 mm) and a UV detector (Kim et al., 1999).
3. Results and discussion
3.1. Central metabolic pathways of A. pullulans CGMCC1234
The activities of the key enzymes involved in the central metabolic pathways of
A. pullulans CGMCC1234 were determined in the cell-free extract. Meawhile,
Pseudomonas sp. No.2120 and Sphingomonas paucimobilis ATCC 31461 were used
as control strains.
As shown in Table 1, the activities of PFK and G6PD existed in A. pullulans
CGMCC1234, whereas KDPG aldolase activity was not detected. PFK, a rate-limiting
enzyme in the EMP, was the most important key enzyme for regulating glycolytic
pathway. G6PD could catalyze the dehydrogenation reaction of glucose-6-phosphate,
the first step and crucial check point of the PPP. KDPG aldolase was a specific
enzyme existed in the ED, it could catalysis of 2-keto-3-deoxy-6-phospho-gluconate
into the glyceraldehyde 3-phosphate and pyruvic acid. In the metabolism of
3-keto-2-deoxy-6-phosphogluconate via the ED. In the metabolism of Sphingomonas
paucimobilis ATCC 31461, glucose was metabolized via the PPP and the ED. We
found that the EMP existed in A. pullulans CGMCC1234 and the activity of PFK was
84 ± 6 nmol·mg protein-1·min-1. Since the specific activity of KDPG aldolase was
found to be zero, we proposed that the ED did not occur in A. pullulans CGMCC1234.
Ac ce pt e
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In addition, the activity of G6PD was 241 ± 8 nmol·mg protein-1·min-1, revealing that
the PPP might also play a vital role. Table 1 The enzyme activity of central metabolic pathways and the resulting presence/absence of
key metabolic pathways in A. pullulans CGMCC1234，Pseudomonas sp. No.2120 and
Sphingomonas paucimobilis ATCC 31461 Enzyme activity (nmol·mg
A. pullulans CGMCC1234
84 ± 6
189 ± 7
Pseudomonas sp. No.2120
125 ± 4
Sphingomonas paucimobilis ATCC
241 ± 8
59 ± 5
Values are given as means ± standard deviation (n=3).
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3.2. Effect of iodoacetic acid on pullulan fermentation Fig. 1 showed the influence of various iodoacetic acid concentrations on pullulan
fermentation. Pullulan declined fast with the iodoacetic acid increased. There was
almost no pullulan production when the concentration of iodoacetic acid over 0.9 mM.
Different from the change of pullulan production, A. pullulans CGMCC1234 cell
growth offered upgrade firstly than descending latter tendency. When the
concentration of iodoacetic acid below 0.6 mM, DCW constantly raised with the
increasing iodoacetic acid. Compared to the control group, the ratio of ATP/ADP of
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0.600 mM iodoaacetic acid group g was llower at diffferent stagees of pullulaan fermentaation
Figg. 1 Influence of iodoacettic acid conccentration on pullulan ferm mentation
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Fig. 2 ATP/ADP ratio at different stages of pullulan fermentation with or without iodoacetic acid.
Previous work had suggested that pullulan synthesis involved UDP-glucose,
required ATP, and proceeded through lipid intermediates (Leathers, 2003). The
reduction of ATP resulted from the inhibition of EMP might lead to the lower pullulan
production. In addition, the decrease of cell biomass could also lead to the decline
production of exopolysaccharide. The PPP was a metabolic pathway parallel to
glycolysis that generated NADPH and pentoses, which could apply for the
biosynthesis of fatty acids, sterols, nucleotides, and amino acid (Kruger, & von
Schaewen, 2003). The PPP did involve oxidation of glucose, its primary role was
anabolic rather than catabolic. Low concentration of iodoacetic acid might suppress
the metabolic flux of the EMP, and then the PPP flux would be promoted. This could
explain why A. pullulans cell grew more under a small amount of iodoacetic acid. Our
observation was consistent with the result of Clark et al. They found that young cells
of A. pullulans catabolized glucose in such a manner that most of the carbon dioxide
came from carbon 1 of glucose (Clark, & Wallace, 1958). However, cell growth fell
with the further increase of iodoacetic acid. The whole process of sugar metabolism
was disrupted by high density of iodoacetic acid, even the mitochondrial respiratory
chain might be broken.
Ac ce pt e
168 169 170
4. Conclusions The existence of the activities of PFK and G6PD indicated that the central 9
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metabolic pathways of A. pullulans included EMP and PPP. Meanwhile, no KDPG
aldolase activity revealed that the ED did not happen in A. pullulans CGMCC1234.
Pullulan synthesis was inhibited by iodoacetic acid, while a small amount of
iodoacetic acid could stimulate cell growth. Low concentration of iodoacetic acid
might impel carbon flux flow toward the PPP, but reduce the flux of the EMP.
This project was supported by the Fundamental Research Funds for the Central
Universities (Program No.2015BQ042).
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The central metabolic pathways of A. pullulans included EMP and PPP, but no ED. Pullulan synthesis was inhibited by iodoacetic acid along with the decrease
of ATP. DCW raised firstly then declined as iodoacetic acid increased.
A few of iodoacetic acid might impel carbon flux flow toward the PPP.
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